Misshapen Enzyme Links Both Rare and Common Forms of ALS

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is one of the most common neurodegenerative disorders, and one of the most puzzling. In a few cases, it is caused by inherited mutations in a gene encoding the enzyme superoxide dismutase 1 (SOD1)— mutations that result in SOD1’s taking an abnormal, apparently toxic shape. But the vast majority of ALS cases are not inherited, and in these cases scientists have been unable to find strong evidence of SOD1’s involvement.

Now, however, researchers report that in these more common, “sporadic” ALS cases, the SOD1 enzyme can indeed become misshapen and toxic, just as it appears to do in the rarer cases caused by SOD1 mutations. The findings suggest that these misshapen, “misfolded” forms of SOD1 should be a target for potential ALS drugs.

“Therapies targeting mutant SOD1 in inherited ALS are already being developed, and now in light of our results, these approaches may be extended to target the misfolded protein in common, sporadic ALS,” says Daryl Bosco, an assistant professor of neurology at the University of Massachussetts Medical School, and lead author of a study published online in Nature Neuroscience on Oct. 17.

Not a red herring after all?

After scientists first linked SOD1 mutations to inherited ALS in 1993, they hoped that this form of ALS would be a good model for the much more common, sporadic ALS cases, which have no obvious genetic component. Research into other neurodegenerative diseases such as Alzheimer’s and Parkinson’s has tended to make progress that way—the rare genetic forms, which are easier to study, providing clues to the causes of the more common sporadic forms.

But the clues provided by inherited ALS haven’t translated very well to sporadic ALS. For a long time, no one could find evidence that SOD1 was malformed or toxic in sporadic ALS cases. Mice were engineered with the mutant SOD1 gene and developed an ALS-like condition, but drugs that seemed to treat them failed to work in human sporadic-ALS patients. Some researchers have come to wonder whether SOD1 has any relevance to ALS at all outside of the rare SOD1-mutant inherited cases. (See “Mouse Models: Handle With Care.”)

In the new study, Bosco and her colleagues, led by senior author Robert Brown, a veteran ALS researcher who chairs the neurology department at the University of Massachussetts Medical School and was also a senior author of the 1993 SOD1 mutation study, used special antibodies that bind to the mutant SOD1 in inherited ALS cases, but not to normal SOD1. Termed C4F6, the mutant-specific antibodies were raised by co-author Jean-Pierre Julien and colleagues at Laval University in Quebec.

These antibodies apparently recognize an abnormal shape on mutant SOD1 enzymes, and Bosco and Brown and their colleagues hypothesized that the antibodies also would detect misfolded SOD1 enzymes if they existed in sporadic ALS.

In a series of experiments, they found that the C4F6 antibodies did indeed recognize abnormally shaped SOD1 in 4 of 9 tissue samples taken from affected spinal regions of sporadic ALS patients (in the other 5 samples the motor neurons which would have contained any SOD1 had completely degenerated). They also found that when they exposed normal SOD1 to hydrogen peroxide—a process that often occurs in cells as a side effect of energy production and can alter a protein’s shape—the oxidized SOD1 became recognizable by the C4F6 antibodies.

In previous work, the group had shown that mutant SOD1 could harm motor neurons by boosting the production of another stress-related enzyme (P38 kinase), thereby shutting down “axonal transport”—the normal traffic of proteins and other cellular components along motor neurons’ often-lengthy output stalks. In this study, they showed that oxidized SOD1 could do the same. Thus, in principle, an oxidation or other modification of SOD1, perhaps during an episode of cellular stress, could give the enzyme not only an abnormal shape but also a toxic function like that found in inherited ALS.

More than one misfolding?

In July, a group of researchers at Umea University in Sweden reported similar findings. Using individual segments of SOD1, they were able to raise new, mutant-SOD1 specific antibodies. They then used these special antibodies to detect misfolded SOD1 in 100 percent of spinal tissue samples from 37 inherited or sporadic ALS patients (compared with a very low percentage in control samples).

The senior author of this study, Thomas Brännström, notes that his group’s antibodies appear able to label misfolded SOD1 in sporadic ALS tissues more consistently than the C4F6 antibodies did, suggesting that there is more than one kind of misfolded SOD1 in sporadic ALS. “If there are several conformational species of misfolded SOD1, the C4F6 antibody might react only with a subset of such species,” he says.

Melanie Leitner, chief scientific officer of Prize4Life in Cambridge, MA, which funds ALS research, says that she considers the new SOD1 findings exciting and provocative, although she cautions, “How common oxidized or misfolded SOD1 is in ALS patients remains an open question.” In the past several years, researchers have consistently found a separate protein, TDP-43, in abnormal aggregates within dying neurons in most inherited and sporadic ALS cases, and partly for this reason some ALS laboratories have shifted their focus away from SOD1. (See “ALS Researchers Focus on Mystery Protein TDP-43”.) If more evidence for SOD1’s role emerges, that could change. There is precedent: Two separate aggregating proteins, amyloid-beta and tau, are known to be involved in Alzheimer’s disease, for example. But “so far I feel kind of agnostic about [the issue of] SOD1 versus TDP-43,” Brown says.

Is ALS another protein aggregation-related disorder?

If misshapen molecules of SOD1 do indeed lie at the heart of sporadic ALS, then treatments could be based on antibodies such as C4F6. Julien and his colleagues have reported separately that SOD1-mutant mice took significantly longer to die of their ALS-like condition after being injected with C4F6 antibodies. “A couple of small startup companies are already developing antibodies as potential therapies against mutant SOD1,” says Brown. He adds that past SOD1-mutant mouse trials may have failed to translate well to human trials not because the model was inadequate but because the candidate drugs had only weak effects.

It is so far unclear what form or species of abnormal SOD1 would be the best target in sporadic cases, even if the protein is involved in driving this more common variant of ALS. When a protein misfolds, it often exposes sticky elements that are normally inaccessible; this makes the protein more likely to cling to other copies of itself. Proteins including A-beta and tau in Alzheimer’s, alpha-synuclein in Parkinson’s, and prion protein in Creutzfeldt-Jakob disease undergo a similar process, which has been linked to oxidation and can spread in a contagion-like manner within tissues. At least several biotechnology companies now aim to treat such diseases by somehow interrupting this aggregation process, particularly in its early stages when the aggregates, though small and soluble, are believed to be particularly toxic. (See “Amyloid Beta ‘Oligomers’ May be Link to Alzheimer’s Dementia”)

To Bosco and Brown, it is too early to tell how relevant the aggregation process will be for ALS. The SOD1 enzyme is larger and structurally more stable than other proteins implicated in aggregation-linked disorders. But so far, they can’t exclude the possibility that small, still-soluble clusters of SOD1 are somehow causing the toxicity: “That’s really our next goal—to further characterize the biochemical and structural properties of these SOD1 proteins that are modified in sporadic ALS cases,” says Bosco.

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